Ebola virus (EBOV) and Marburg virus (MARV) are highly lethal hemorrhagic fever viruses of the Filoviridae family of viruses. Currently, there are no effective oral or easily scalable therapies for either virus. Both EBOV and MARV manifest vigorous anti-host evasion strategies focused on blocking the host antiviral interferon response. Both viruses interfere with host cell sensing of infection via their structural protein VP35, which inhibits viral dsRNA sensing and triggering of the host antiviral response. Therapeutic strategies that enhance host pathways that are inhibited by these viruses could therefore be of significant value. We have discovered that the well-tolerated FDA-approved oral anti-parasitic drug nitazoxanide (NTZ) inhibits infectious EBOV replication in human A549 cells in vitro. Furthermore, we find that NTZ enhances the RIG-I-like receptor (RLR) proteins retinoic-acid-inducible protein I (RIG- I) and Melanoma Differentiation-Associated protein 5 (MDA5), their common adaptor protein mitochondrial antiviral signaling protein (MAVS), the IFN-inducible double-stranded (ds) RNA sensor protein kinase R (PKR), and interferon signaling pathways. In addition, using deadCas9/CRISPR editing, we ablated RIG-I and PKR in A549 cells, and found that EBOV growth was significantly increased and in parallel NTZ's inhibitory effects were significantly attenuated. These results thus indicate that RIG-I and PKR are both required and non-redundant host restriction factors for EBOV. Moreover, PKR and RIG-I are both targeted by and necessary for NTZ's action indicating that NTZ counteracts the EBOV VP35 protein's ability to block RIG-I and PKR sensing of EBOV infection. In Aim 1 of this proposal, we will test the hypothesis that NTZ also inhibits MARV in vitro, which we strongly expect given the importance of MARV's VP-35 protein in its pathogenesis. In Aim 1, we will also test our hypothesis that NTZ inhibits both EBOV and MARV in vivo in the guinea pig model. In Aim 2, we will test the impact of MDA5 or MAVS inhibition, in addition to studying RIG-I, PKR or GADD34 depletion on MARV replication. We will compare these results to experiments evaluating EBOV replication in monocytic THP-1 cells. These experiments will evaluate the role of these discrete sensors in NTZ's inhibitory effects upon EBOV and MARV and it will test the hypothesis that NTZ works through PKR and RIG-I in inhibition of MARV as it does in EBOV infection. Building on these studies, using an unbiased RNA-seq approach, we will determine transcriptomic signatures of cellular perturbations induced by NTZ in the setting of EBOV and MARV infection allowing us to define host immune regulatory circuits involved in cytoplasmic sensing of EBOV and MARV and the impact of NTZ. We anticipate that these studies will provide scientific underpinning for repurposing NTZ as a therapy for EBOV and MARV and uncover key innate immune molecules that control their pathogenesis.